New techniques recently developed in this laboratory for imaging freeze-dried cells and molecules in the electron microscope will be used to visualize the three crucial steps that are involved in membrane circulation in living cells: (l) vesicle formation from "donor" compartments, (2) vesicle translocation through the cytoplasm, and (3) vesicle fusion with "acceptor" compartments. Vesicle formation will be studied in macrophages and reticulocytes (immature erythrocytes), vesicle translocation in elongated nerves and pigment cells, and vesicle fusion in certain specialized secretory or exocytotic cells. The aim will be to visualize the structural specializations that allow the cell to shuttle material through a series of fluid membrane compartments without losing the physical and biochemical individuality of each compartment. Particular effort will be directed toward improving the methods of quick-freezing, freeze-etching, and rotary-replication with platinum which will be used to prepare our samples for this new type of electron microscopy. Also efforts will be made to improve current methods for extracting soluble components from living cells, so that broader interior views can be obtained, and to improve methods of immunocytochemistry, so that antibody "decoration" of specific structures can be witnessed directly in the electron microscope, without needing to resort to the indirect methods used currently. Information thus gained on the organization and chemical identity of the structures outlined above will help to explain how the cell maintains such a rich and varied cross-traffic between various of its organelles, how it takes up and processes materials that it picks up from its environment, and how it discharges into its environment the materials that it manufactures.